Abnormal glycosylation is a well known attribute of cancer cells. (1-4) A number of specific glycan structures serve as biomarkers of tumor growth. Of particular significance are the sialic acids, of which more than 40 naturally occurring derivatives have been identified. Sialic acids are nine-carbon carboxylated sugars which exist in three primary forms. The most common is N-acetyl-neuraminic acid (2-keto-5-acetamido-3,5-dideoxy-D-glyc-ero-D-galactononulopyranos-1-onic acid (often abbreviated as NeuSAc, NeuAc, or NANA). A second common form is N-glycolyl-neuraminic acid (Neu5Gc or NeuGc), in which the N-acetyl group of NeuAc is hydroxylated. A third primary sialic acid is 2-keto-3-deoxy-nonulosonic acid (KDN).
Typically found at the reducing end of glycans attached to cell surfaces or plasma proteins, sialic acids are typically over expressed in tumor cells, relative to normal tissues. These terminal sialic acids are involved in cellular adhesion and are components of cell surface receptors. Excess sialylation may mask specific cellular recognition sites, which is an important component of physiological responses to cancer cells. Lewis X and Lewis A blood group antigens, which are sialic acid containing proteins, are also typically overexpressed in carcinomas. Additional qualitative and quantitative changes in tumor cell surface sialic acids are associated with progression to malignancy. Tumor cells can change the sialo-glyco-conjugates expressed on their plasma membranes, which affects their ability to invade. (5-16) Quantitative and qualitative assessment of protein sialylation in biological samples is increasingly recognized as a valuable contribution to diagnosis, prognosis and monitoring of conditions associated with over-sialylation of proteins. Such conditions include diabetes and myeloma, epithelial, breast, ovarian, oral, gastrointestinal, prostate, endometrial, lung, colon, pancreatic, and thyroid cancers. Assessments of protein sialylation are also useful in diagnosis, prognosis and monitoring of conditions associated with under-sialylation of proteins, including HIV-1 infection, cystic fibrosis, hereditary inclusion body myopathy (HIBM), Henoch-Schonlein purpura, and IgA nephropathy. (17-27)
Changes in sialylation status associated with disease conditions often involve low abundance proteins. Accordingly, an initial purification of sialic acid containing proteinsis required for analysis of sialylation status. Methods for isolation of sialylated proteins thus provide means of qualitative and quantitative assessment of sialylation status as well as means of identifying specific biomarkers of conditions associated with a change of sialylation status. A variety of techniques for purification of sialylated proteins have been reported. WO2005/107491 teaches a lectin affinity chromatography. EP 1 428 878 teaches an anion exchange method. JP 08027181 teaches use of a chitosan-immobilized stationary phase. A variety of HPLC and hydrophilic interaction chromatography techniques have also been reported. These techniques generally require expensive reagents or relatively cumbersome procedures or utilize buffers that are not suited for mass spectrometry studies.
Accordingly, the present invention provides a simple, convenient method for isolation of sialylated peptides that is particularly suited for mass spectrometry analysis. The method generally utilizes titanium dioxide or zirconium dioxide chromatographic stationary phase material and a loading buffer comprising a substituted aromatic carboxylic acid, preferably dihydroxy benzoate or phthalic acid. The invention further provides methods of identifying biomarkers of a condition associated with change of sialylation status as well as methods for diagnosing, prognosing or monitoring such conditions. In other embodiments, the method is also useful for isolation and analysis of phosphorylated peptides.